The invention relates to nucleoside derivatives that inhibitor HCV replicon RNA replication. In particular, the invention is concerned with the use of phosphoramidate esters of 4′-substituted nucleosides that inhibit subgenomic HCV RNA replication and pharmaceutical compositions containing said compounds.
Hepatitis C virus is the leading cause of chronic liver disease throughout the world (Boyer, N. et al. J. Hepatol. 2000 32:98-112). Patients infected with HCV are at risk of developing cirrhosis of the liver and subsequent hepatocellular carcinoma and hence HCV is the major indication for liver transplantation.
HCV has been classified as a member of the virus family Flaviviridae that includes the genera flaviviruses, pestiviruses, and hapaceiviruses which includes hepatitis C viruses (Rice, C. M., Flaviviridae. The viruses and their replication. In: Fields Virology, Editors: B. N. Fields, D. M. Knipe and P. M. Howley, Lippincott-Raven Publishers, Philadelphia, Pa., Chapter 30, 931-959, 1996). HCV is an enveloped virus containing a positive-sense single-stranded RNA genome of approximately 9.4 kb. The viral genome consists of a 5′ untranslated region (UTR), a long open reading frame encoding a polyprotein precursor of—approximately 3011 amino acids, and a short 3′ UTR. The 5′ UTR is the most highly conserved part of the HCV genome and is important for the initiation and control of polyprotein translation.
Genetic analysis of HCV has identified six main genotypes which diverge by over 30% of the DNA sequence. More than 30 subtypes have been distinguished. In the US approximately 70% of infected individuals have Type 1a and 1b infection. Type 1b is the most prevalent subtype in Asia (X. Forns and J. Bukh, Clinics in Liver Disease 1999 3:693-716; J. Bukh et al., Semin. Liv. Dis. 1995 15:41-63).
Unfortunately Type 1 infectious is more resistant to therapy than either type 2 or 3 genotypes (N. N. Zein, Clin. Microbiol. Rev., 2000 13:223-235).
Viral structural proteins include a nucleocapsid core protein (C) and two envelope glycoproteins, E1 and E2. HCV also encodes two proteases, a zinc-dependent metalloproteinase encoded by the NS2-NS3 region and a serine protease encoded in the NS3 region. These proteases are required for cleavage of specific regions of the precursor polyprotein into mature peptides. The carboxyl half of nonstructural protein 5, NS5B, contains the RNA-dependent RNA polymerase. The function of the remaining nonstructural proteins, NS4A and NS4B, and that of NS5A (the amino-terminal half of nonstructural protein 5) remain unknown. It is believed that most of the non-structural proteins encoded by the HCV RNA genome are involved in RNA replication
Currently there are a limited number of approved therapies available for the treatment of HCV infection. Existing therapies and new therapies currently in development for treating HCV and inhibition of HCV NS5B polymerase have been reviewed: R. G. Gish, Sem. Liver. Dis., 1999 19:5; Di Besceglie, A. M. and Bacon, B. R., Scientific American, October: 1999 80-85; G. Lake-Bakaar, Current and Future Therapy for Chronic Hepatitis C Virus Liver Disease, Curr. Drug Targ. Infect. Dis. 2003 3(3):247-253; P. Hoffmann et al., Recent patents on experimental therapy for hepatitis C virus infection (1999-2002), Exp. Opin. Ther. Patents 2003 13(11):1707-1723; M. P. Walker et al., Promising Candidates for the treatment of chronic hepatitis C, Exp. Opin. Investing. Drugs 2003 12(8):1269-1280; S.-L. Tan et al., Hepatitis C Therapeutics: Current Status and Emerging Strategies, Nature Rev. Drug Discov. 2002 1:867-881; J. Z. Wu and Z. Hong, Targeting NS5B RNA-Dependent RNA Polymerase for Anti-HCV Chemotherapy, Curr. Drug Targ.-Infect. Dis. 2003 3(3):207-219. The development of resistance by HCV strains along with existing strains which are refractive to current therapy make new anti-HCV compounds very desirable.
A number of potential molecular targets for drug development as anti-HCV therapeutics have now been identified including, but not limited to, the NS2-NS3 autoprotease, the N3 protease, the N3 helicase and the NS5B polymerase. The RNA-dependent RNA polymerase is absolutely essential for replication of the single-stranded, positive sense, RNA genome. Consequently, this enzyme has elicited significant interest among medicinal chemists. Nucleoside inhibitors of RNA polymerase can act either as a chain terminator during DNA synthesis or as a competitive inhibitor which interferes with nucleotide binding to the polymerase. To function as a chain terminator the nucleoside analog must be taken up be the cell and converted in vivo to a triphosphate to compete for the polymerase nucleotide binding site. The required conversion of nucleosides to the corresponding triphosphate is commonly mediated by cellular kinases imparting additional structural requirements on a potential nucleoside polymerase inhibitor. This also limits the direct evaluation of nucleosides as inhibitors of HCV replication to cell-based assays. Modification of the furanose ring of nucleosides has afforded compounds with anti-viral activity. Modification of the 2′- and 3′-positions of the sugar ring has been extensively investigated. Modification of the 4′-position of the furanose ring has been explored to a lesser extent because of the difficulties associated with introduction of substituents at this position.
Maag et al. (J. Med. Chem. 1992 3 5:1440-1451) disclose the synthesis of 4′-azido-2-deoxyribonucleosides and 4-azido nucleosides. C. O'Yang et al. (Tetrahedron Lett. 1992 33(1):37-40 and 33(1):41-44) disclose the synthesis 4′-cyano, 4′-hydroxymethyl- and 4′-formyl substituted nucleosides. These compounds were evaluated as anti-HIV compounds.
In WO02/100415 published Dec. 19, 2002 (US 2003/0236216 A1), R. R. Devos et al. disclose 4′-substituted nucleoside compounds that exhibit anti-HCV activity. Four compounds explicitly identified include the 4′-azido compound, 1a, the 4′-ethynyl compound 1b, the 4′-ethoxy compound 1c and the 4′-acetyl compound 1d. Other exemplified modifications of the ribose moiety exemplified include the 2′-deoxy 2a derivative, 3′-deoxy derivative 2b, the 3′-methoxy derivative 2e, the 3′-fluoro derivative 2c and the 2′,3′-difluoro derivative 2d. In WO2004/046159 published Jun. 3, 2004 (now U.S. Pat. No. 6,846,810), J. A. Martin et al. disclose mono-, di-, tri- and tetra-acyl prodrugs of 1a useful for treating HCV-mediated diseases. Both U.S. applications are hereby incorporated by reference in their entirety.
Y.-H. Yun et al. (Arch. Pharm. Res. 1985 18(5):364-35) disclose the synthesis and antiviral activity of 4′-azido-2′-deoxy-2′-fluoro-arabinofuranosyl nucleosides (3: R=H, Me and Cl).

G. S. Jeon and V. Nair (Tetrahedron 1996 52(39): 12643-50) disclose the synthesis 4′-azidomethyl-2′,3′-deoxyribonucleosides 4 (B=adenine, thymine and uracil) as HIV reverse transcriptase inhibitors.
I. Sugimoto et al. (Bioorg. Med. Chem. Lett. 1999 9:385-88) disclosed the synthesis and the HIV and H. simplex bioassay of 4′-ethynyl-2′-deoxycytidine (5) and other two-carbon substituents at the 4′-position. T. Wada et al. (Nucleosides & Nucleotides 1996 15 (1-3):287-304) disclose the synthesis and anti-HIV activity of 4′-C-methyl nucleosides.
In WO02/18404 published Mar. 7, 2002, R. Devos et al. disclose novel and known purine and pyrimidine nucleoside derivatives and their use as inhibitors of subgenomic HCV replication and pharmaceutical compositions containing said nucleoside derivatives. The compounds disclosed consist of nucleosides with substituted purine and pyrimidine bases.

H. Ohrui et al. (Antimicrobial Agents and Chemother. 2001 45(5):1539-1546; see also S. Koghgo et al., Tennen Yuki Kagobutsu Toronkai Koen Yoshishu 2000 42:835 (Chem. Abs. 2001:102156 and H. Ohrui et al. WO2000069876 published Nov. 23, 2000) disclose the synthesis and anti-HIV activity of 4′-C-ethynyl-β-D-arabino- and 4′-C-ethynyl-2′-deoxy-β-D-ribo-pentofuranosyl pyrimidines and -purines. 4-Ethynyl-cytarabine (6a) exhibits good anti-HIV activity while the corresponding nucleoside wherein the base was thymine 6b was inactive. Several 4′-C-ethylnyl-2′-deoxy-β-D-ribo-pentofuranosyl pyrimidines and -purines were potent inhibitors of HIV reverse transcriptase (HIV-RT).
K. Kitano et al. (Tetrahedron 1997 53(39):13315-13322) disclose the synthesis 4′-fluoromethyl 2-deoxy-D-erythro-, ribo- and arabino-pentofuranosyl cytosines and anti-neoplastic activity.
4′-Azidocytidine, 4′-azidouridine, 4′-ethynylcytidine, 4′-ethynyluridine, 4′azido-arabinose (see e.g. U.S. Ser. No. 60/603,778 which is incorporate by reference in its entirety), 4′-(Z-2-chlorovinyl)cytidine and 4′-(Z-2-chlorovinyl)uridine have exhibited activity against HCV and Flaviviridiae in cell culture or phosphorylated analogs were active against HCV polymerase in vitro. However, more potent compounds are desirable to provide safe therapeutically effective levels in vivo. Surprisingly, certain phosphoramidate derivatives have now been found to exhibit useful biological activity against Flaviviridae.
Although nucleoside derivatives have proven to be effective inhibitors of HCV polymerase, their practical utility is often limited by two factors. Firstly, suboptimal physical properties and poor pharmacokinetics frequently limit the intracellular concentration of the nucleoside derivative. The present invention relates to phosphoramidate derivatives of 4′-substituted nucleosides compounds with improved physiochemical and pharmacokinetic properties. These derivatives more efficiently permeate the intestinal mucosa and ultimately are transported into the cell. These “pronucleotides” enhance biological activity, bioavailability or stability of the parent nucleotide (for reviews, see e.g., R. J. Jones and N. Bischofberger, Antiviral Res. 1995 27; 1- 15 and C. R. Wagner et al., Med. Res. Rev. 2000 20:417-451).
Secondly, if the prodrug successfully penetrates an infected cell and is converted to the parent nucleoside, the biologically activity of these compounds depends upon kinase-mediated phorsphorylation to generate the nucleoside triphosphate. Chemically modified nucleosides that are effective enzyme inhibitors are frequently poor substrates for endogenous nucleoside kinases resulting in the inefficient product of the triphosphate. Furthermore, cells with low levels of nucleoside kinases are unable to phosphorylate the nucleoside analog. Formation of the monophosphate by a nucleoside kinase is normally rate-limiting and the second and third phosphorylations are less sensitive to modifications to the nucleoside.
                R5 is H or a substituent        R6 is cytidine, uridine or a 5-substituted derivative thereof        
Aryloxy phosphoramidate derivatives 7a afford a mechanism to overcome both problems. The phosphate moiety is masked with neutral lipophilic groups to obtain a suitable partition coefficient to optimize uptake and transport into the cell. Enzyme-mediated hydrolysis of the ester produces a nucleoside monophosphate 7e wherein the rare limiting initial phosphorylation is unnecessary. (Scheme I) C. McGuigan et al., Antiviral Res. 1992 17(4):311-321; Antiviral. Res. 1991 15:255-263; J. Med. Chem. 1993 36(9):1048-1052; Antiviral Res. 1994 24:69-77; J. Med. Chem. 1996 39:1748-1753; Bioorg. Med. Chem. Lett. 1996 6:2359-2361; P. Franchetti et al., J. Med. Chem. 1994 37:3534-3541; G. Valette et al., J. Med. Chem. 1996 39:1981-1990; J. Balzarini et al., FEBS Lett. 1997 410: 324-328; D. Saboulard et al., Mol. Pharmacol. 1999 56:693-704; A. D. Siddiqui et al., Bioorg. Med. Chem. Lett. 1999 9:2555-2260; S. C. Tobias and R. F. Borch J. Med. Chem. 2001 44:4475-4480; K. S. Gudmundsson et al., Nucleosides, Nucleotides & Nucleic Acids 2003 22(10):1953-1961; D. Siccardi et al., Eur. J Pharm. Sci. 2004 22:25-31.
Phosphoramidate diesters of nucleoside compounds have been reported including AZT (zidovudine), d4T (stauvidine), FudR (5-fluorodeoxyuridine), 2′-deoxyuridine, thymidine, d4A (2′,3′-didedehydro-2′,3′-dideoxyadenosine), isoddA (2′,3′-dideoxy-3′-oxoadensoine), FLT (alovudine, 3-deoxy-3-fluorothymidine), ddC (2′,3′-dideoxycytosine), ddA (dideoxyadenosine), hypoxallene, 2′,3′-dideoxy-3′-thiacytidine (3TC) and Ara-C (Wagner, id., p. 438).